LID scientists are collaborating with scientists from Medimmune under a CRADA to generate candidate vaccines against avian influenza viruses of each subtype, including H5N1 viruses that have caused human infections since 2003, as well as H6 and H7 viruses. The vaccines were generated using plasmid based reverse genetics and each contains the hemagglutinin and neuraminidase genes from an avian influenza virus and six internal gene segments from the AA ca virus. Based on promising preclinical data in mice and ferrets, clinical lots of H5N1 and H7N3 ca vaccines were generated and Phase I clinical trials of the safety and immunogenicity of the vaccines for healthy adults were undertaken under an IND. [unreadable] An H6 influenza virus was identified as a potential progenitor of the H5N1 viruses that emerged in Hong Kong in 1997 because the two viruses shared high sequence homology in 7 gene segments. This virus continues to circulate in the bird population in Asia, and other H6 viruses are prevalent in birds in North America and Asia. H6 was the most abundantly detected influenza virus subtype in wild birds from Europe and the Americas. The prevalence of H6 viruses in aquatic and terrestrial birds increases the potential for transmission to humans and therefore this subtype may pose a pandemic risk. [unreadable] We evaluated the antigenic and genetic relatedness of 14 H6 influenza viruses, and their ability to replicate and induce a cross-reactive immune response in mice and ferrets. The H6 viruses replicated to different levels in the respiratory tract of mice and ferrets, causing varying degrees of morbidity and mortality in these two models demonstrating that mammals can be productively infected with H6 viruses. Eleven of the 14 viruses replicated in the lungs and 10 replicated in the NT of mice without prior adaptation. Five viruses that replicated to varying levels in mice were evaluated in ferrets and 4 of them replicated in the respiratory tract of ferrets. Viruses that replicated to moderate to high titers in the lungs induced high neutralizing antibody titers in the sera. Neutralizing antibody titers detected in the sera of ferrets generally correlated with those in mice, but in neither model did the immunogenicity correlate fully with the ability of the viruses to replicate in the respiratory tract. The H6 viruses elicited poorly cross-reactive neutralizing antibodies in ferrets. [unreadable] Based on the genetic analysis and antigenic cross-reactivity of post-infection sera of mice and ferrets infected with H6 viruses isolated from different continents, three H6 viruses, A/duck/Hong Kong/182/77 (H6N9), A/mallard/Alberta/89/85 (H6N2) and A/teal/HK/W312/97 (H6N1), were selected for vaccine development. We generated three live attenuated H6 vaccine candidates that contained the HA and NA gene segments from the wt H6 viruses and the six internal protein gene segments from the AA ca vaccine donor virus. All three H6 vaccine candidates exhibited phenotypic properties of ts, ca and att conferred by the internal gene segments from the AA ca virus. Intranasal administration of a single dose of the three H6 ca vaccine viruses induced neutralizing antibody in mice and ferrets. A single dose of each H6 ca vaccine conferred significant protection from replication of homologous wt virus in the respiratory tract in mice and ferrets; protection from heterologous virus challenge was variable. The A/teal/HK/W312/97 ca virus provided the broadest cross-protection against challenge with three antigenically distinct H6 wt viruses. These findings provide support for clinical evaluation of this H6 ca vaccine virus. An IND application for the phase I evaluation of this vaccine was approved by the FDA and a clinical trial is planned.[unreadable] In addition to the development and evaluation of vaccines against avian influenza viruses, we have investigated the prophylactic and therapeutic efficacy of human monoclonal antibodies against H5N1 influenza. In collaboration with investigators from Vietnam and Switzerland, we generated neutralizing anti-H5N1 human monoclonal antibodies (mAbs) and tested their efficacy for prophylaxis and therapy in a murine model of infection. In vivo, the mAbs conferred protection from lethality in mice challenged with A/Viet Nam/1203/04 (H5N1) in a dose-dependent manner. mAb prophylaxis provided a statistically significant reduction in pulmonary virus titer, reduced associated inflammation in the lungs, and restricted extrapulmonary dissemination of the virus. Therapeutic doses of 3 mAbs provided robust protection from lethality at least up to 72 h post-infection with A/Viet Nam/1203/04 (H5N1) and were also therapeutically active in vivo against the A/Indonesia/5/2005 (H5N1), a virus that belongs to a different genetic clade. These studies provide proof of concept that fully human mAbs with neutralizing activity can be rapidly generated from the peripheral blood of convalescent patients and that these mAbs are effective for the prevention and treatment of H5N1 infection in a mouse model. A panel of neutralizing, cross-reactive mAbs might be useful for prophylaxis or adjunctive treatment of human cases of H5N1 influenza. We are actively involved in mapping the epitopes recognized by these and additional mAbs.